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. Author manuscript; available in PMC: 2021 Jul 21.
Published in final edited form as: Cancer Res. 2020 Apr 7;80(11):2125–2137. doi: 10.1158/0008-5472.CAN-19-3018

Figure 2. PADI4 suppresses pro-tumorigenic biological responses in the tumor cell.

Figure 2.

(A) Effects of PADI4 knockdown on biological responses of MCF10Ca1h cells in vitro. (B, C) Effect of PADI4 knockdown on tumorsphere formation in multiple breast cancer cells lines (B), with representative images shown for MDA-MB-231 LM2 (C). (D-F) Effect of 3-day pharmacological inhibition of PADI4 by GSK484 on H3 citrullination in MCF10Ca1h cells (D), tumorsphere formation assay in MCF10Ca1h cells and MCF7E cells (E), and invasion and migration in MCF10Ca1h cells (F). GSK106 is the inactive control compound. (G) Western blots for overexpression of PADI4 isoforms in total cell lysates from MCF10Ca1h cells. FL, full-length; X1, X2, X3, X7, variants. For details of isoforms, see Supplementary Fig. S2. EV, Empty vector. β-Actin, loading control. (H) Quantification of nuclear PADI4 protein by Western blot, normalized to Lamin B1. (I) Quantification of PADI4 citrullination activity following PADI4 isoform overexpression in MCF10Ca1h cells. Cells treated with vehicle or 17-β-estradiol (E2) and calcium ionophore (A23187) were analyzed by Western blotting with H3R2/8/17citr antibodies and normalized to Lamin B1. (J) Effect of PADI4 overexpression on tumorsphere formation in MCF10Ca1h cells. Results are mean +/− SD for n=3. Statistics are Dunnett’s multiple comparison test (A-B, J) with shCON or EV as the comparators; and unpaired t-test in D-E. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.